Project Summary: Predictable mammalian cell engineering is essential both for advancing quantitative biology and realizing the promise of precision medicine. In order to develop a predictive framework for cell engineering, we need to understand how gene regulation functions in the context of a dynamic chromatin environment. While the last few years have witnessed a boom in genetic and epigenetic editing tools, there are still major challenges for measuring and predicting the effect of chromatin on gene expression. First, chromatin- mediated control results in cell-to-cell heterogeneity of gene expression, so many questions are difficult to answer with methods that average across cells. Second, in most experimental designs the chromatin state and DNA composition vary at the same time, making it difficult to disentangle the role of chromatin alone. Third, chromatin regulation spans multiple molecular mechanisms and length-scales, making it difficult to integrate these data into a coherent predictive model. In order to address these challenges, we will develop and combine new tools in synthetic biology, single-cell techniques, and mathematical modelling. We will directly manipulate the chromatin state via recruitment and release of chromatin regulators at a defined locus, and then measure the outcome in single cells over time using time-lapse microscopy, flow cytometry and next generation sequencing. Finally, we will build a mathematical model that integrates molecular details at a specific locus with the overall chromatin state in the same cells. We will use these tools to answer essential questions about the role of chromatin dynamics on gene regulation: (1) Why do different cell types have different silencing dynamics and epigenetic memory? (2) How fast and how far do chromatin modifications and their effects spread? (3) How do transcription factors at promoters and enhancers interact with chromatin regulators to determine gene expression? Together, the answer to these basic questions and the development of new techniques for measuring, controlling, and modeling gene expression will advance both mammalian synthetic biology and the basic biology fields of chromatin and gene regulation.